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Novel scintillating nanocomposite for X-ray induced photodynamic therapy
Radiation Measurements 121 (2019) 13–17
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Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Novel scintillating nanocomposite for X-ray induced photodynamic therapy Lenka Procházková a b c
, Iveta Terezie Pelikánová , Eva Mihóková , Roman Dědic , Václav Čuba
a,b
b
a,∗
c
T
b
Institute of Physics of the Czech Academy Od Sciences, Cukrovarnická 10, 162 53, Prague, Czech Republic Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7, 115 19, Prague, Czech Republic Charles University, Faculty of Math and Physics, Ke Karlovu 3, 121 16, Prague, Czech Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanocomposite Photodynamic therapy Singlet oxygen Zinc oxide Scintillator
We synthesize a novel prospective nanocomposite material for X-ray induced photodynamic therapy. ZnO:Ga core is coated by the SiO2 shell facilitating a functionalization by a photosensitizer, protoporphyrin PpIX layer. By steady-state and time resolved spectroscopy we confirm the presence of energy transfer between the core and the photosensitizer, an essential requirement to induce a cytotoxic effect. Using the commercial chemical probe we also demonstrate the ability of prepared ZnO:Ga@SiO2-PpIX nanocomposite to generate singlet oxygen. Presented results show that this nanocomposite can be considered as a good candidate for potential application in X-ray induced photodynamic therapy.
1. Introduction Photodynamic therapy (PDT) is treatment in which visible or nearinfrared light is used to activate a photosensitizer (PS), administered to target a diseased tissue of interest. In the presence of ground state oxygen, the light-activated PS creates reactive oxygen species, especially highly cytotoxic singlet oxygen, that can induce tissue death (Henderson and Dougherty, 1992; Agostinis et al., 2011). PDT was developed primarily for treatment of cancer and precancers. Due to limited depth of light penetration, PDT is most commonly used to treat superficial lesions, like cancer or precancer of the skin (MacCormack, 2006). To overcome the problem of reaching deeply residing tumors in conventional PDT, several approaches have been developed. One or more optical fibers for light delivery are inserted into the target tissue (Shafirstein et al., 2017). Other approaches use specific types of nanoparticles to which PS is attached. Two-photon excitation PDT exploits upconversion nanoparticles that are excited by two-photon absorption of near infrared light and emit higher energy photons to activate PS molecules (Yang et al., 2015; Park et al., 2015; Idris et al., 2015; Chen et al., 2015; Shanmugam et al., 2014; Zhou et al., 2012). In SelfLighting PDT (Chen, 2008; Zhang et al., 2008) the light is generated by afterglow nanoparticles with attached photosensitizers. When the nanoparticle-photosensitizer conjugates are targeted to tumor, the light from afterglow nanoparticles will activate the photosensitizers. Therefore, no external light is required for treatment. X-ray induced PDT (PDTX) exploits X-ray excited photosensitizers (Ma et al., 2014; Liu et al., 2017), quantum dots (Juzenas et al., 2008)
∗
or scintillating nanoparticles (Chen and Zhang, 2006; Wang et al., 2016). PDTX is a combination of radiotherapy and PDT providing more efficient tumor cell killing than the radiotherapy alone. After the X-ray energy is absorbed by a scintillating nanoparticle, it is either radiatively or nonradiatively transferred to the PS molecule. To achieve a cytotoxic effect at therapeutic doses, several parameters, such as light yield of a scintillating nanoparticle, efficiency of energy transfer to PS and cellular uptake of nanoparticles have to be well optimized (Morgan et al., 2009). The nanoparticle uptake by tumor blood vessels can be improved by enhanced permeability and retention (EPR) effect promoted, among others, by suitable nanoparticle size of about 50 nm (Maeda et al., 2013). Various nanocomposite systems have already been shown as potential candidates for PDTX treatment. These systems were based on PS molecule attached to a scintillating core, such as Y2O3 (Scaffidi et al., 2011), Tb2O3 (Bulin et al., 2013), LaF3:Ce3+ (Zou et al., 2014), CeF3:Tb3+ (Popovich et al., 2016), LuAG:Pr3+ (Popovich et al., 2018) or ZnO (Liu et al., 2008). ZnO is a known scintillator with ultrafast, subnanosecond excitonic decay. It is a semiconductor with a wide band gap of 3.4 eV. Its extremely high exciton binding energy of about 60 meV ensures efficient luminescence at room temperature (RT). ZnO in various forms has been studied due to a number of applications especially in optoelectronics. Controlled doping with Ga3+ ions together with heat treatment in reducing atmosphere suppress the slow defect emission around 500 nm and enhances the excitonic emission around 400 nm (Procházková et al., 2015). Therefore, ZnO:Ga3+ was recently also studied for applications requiring fast timing (Turtos et al., 2016), such as time-of
Corresponding author. E-mail address: [email protected] (E. Mihóková).
https://doi.org/10.1016/j.radmeas.2018.12.008 Received 15 October 2018; Received in revised form 11 December 2018; Accepted 14 December 2018 Available online 17 December 2018 1350-4487/ © 2018 Elsevier Ltd. All rights reserved.
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flight positron emission tomography (Karp et al., 2008; Jones and Townsend, 2017; Gundacker et al., 2016). In this work we design a novel nanocomposite system as a candidate for PDTX application. The nanocomposite consists of the ZnO:Ga scintillating core coated by the SiO2 shell facilitating its conjugation to protoporphyrin IX (PpIX) serving as a photosensitizer. We synthesize this system, study its luminescence and scintillation properties to test the energy transfer between the ZnO:Ga3+ core and PpIX outer layer and we also show its ability to generate the singlet oxygen. 2. Materials and methods 2.1. Nanocomposite synthesis ZnO:Ga core was synthesized by photo-induced method (Bárta et al., 2012) using the following chemicals: zinc oxide, gallium nitrate, (all chemicals from Aldrich, 99.999% trace metals basis), formic acid, nitric acid (Penta, p.a.) and hydrogen peroxide (Penta, p.a.). After the irradiation of aqueous solutions, formed solid zinc peroxide was separated and decomposed at 250 °C in air. Two-step annealing followed: annealing at 1000 °C in air performed in the Clasic 0415 VAC vacuum furnace and additional annealing at 800 °C using the Setaram LabSys Evo thermoanalyzer under the Ar/H2 (20:1) atmosphere. The prepared ZnO:Ga nanopowder was coated by a silica layer using a modified sol-gel process described in (Liu et al., 1998), for more details, see (Popovich et al., 2018). Obtained silica-coated ZnO:Ga nanoparticles were conjugated with PpIX using a modified functionalization procedure reported in (Nowostawska et al., 2011), for more details, see (Popovich et al., 2018).
Fig. 1. Diffraction patterns of synthesized nanoparticles (indicated in the figure) compared with the standard dat of ZnO:Ga from ICDD PDF-2 database (card No. 01-073-1368, dashed lines in the figure). Data for ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX are vertically shifted for better visualization.
concentration c (Fe3+). The radiation dose rate D is calculated from c (Fe3+) - c0(Fe3+) = ρ × G (Fe3+) × D, where ρ = 1.024 kg/m3. The obtained value 34 Gy/h for X-ray source used reflects the dose absorbed by the solutions in plastic cuvettes, which may slightly differ in the case of suspensions used in real experiments. 3. Results The diffraction patterns of all synthesized materials are shown in Fig. 1. They are all in agreement with that of ZnO:Ga standard (dashed lines, card No. 01-073-1368) from ICDD PDF-2 database. Prepared nanoparticles correspond to hexagonal crystal structure of wurtzite. The average crystallite size is about 300 nm. This size was determined by application of Halder-Wagner method using FWHM (full width at half maximum) of peaks and the Scherrer constant K = 0.94. The absence of additional peaks in diffraction patterns excludes the presence of any other crystalline phase. Furthermore, it confirms that coating of ZnO:Ga nanoparticle by SiO2 layer and subsequent functionalization preserve the crystal structure of the nanoparticle as well as the fact that SiO2 layer is amorphous. In Fig. 2 we present SEM images of all prepared nanoparticles. The images show ZnO:Ga grains (A), ZnO:Ga modified by the surface SiO2 layer (B) and further functionalized by PpIX (C). As mentioned above, cytotoxic effects of prepared functionalized nanoparticles ZnO:Ga@SiO2-PpIX can only be achieved if there exists an efficient energy transfer between the ZnO:Ga core and PpIX outer layer. A prerequisite for such energy transfer, either radiative or nonradiative, is an overlap between the absorption spectrum of PpIX and RL spectrum of ZnO:Ga. In Fig. 3a we show absorption spectrum of PpIX featuring a strong absorption band peaking around 400 nm, so called Soret band, and weaker Q-bands peaking between 500 and 650 nm. Typical PL spectrum of PpIX under excitation into the Soret band is also shown. Its two bands are peaking at about 635 nm and 705 nm. The peak around 635 nm is due to zero-phonon Q (0,0) emission of monomeric PpIX (Hope et al., 2016; Scolaro et al., 2002). The 705 nm peak occurs via Q (0,1) transition known as J-band, which is also associated with the result of formation of J-aggregates (Villari et al., 2012) when the porphyrin molecules self-aggregate (Hope et al., 2016; Scolaro et al., 2002). Comparison to PpIX absorption with the ZnO:Ga RL spectrum displayed in Figs. 4 and 9 confirms a significant overlap between the Soret band of PpIX and ZnO:Ga RL spectrum. Radioluminescence spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@ SiO2-PpIX are presented in Fig. 4. ZnO:Ga nanoparticles feature an intense emission band around 390 nm attributed to emission of MottWannier exciton formed under excitation of the ZnO host lattice. One
2.2. Characterization methods The XRD measurements were performed using Rigaku MiniFlex 600 diffractometer equipped with Cu X-ray tube (average wavelength Kα1,20.15418 nm). High voltage and current settings used were 40 kV and 15 mA, respectively. The measurement was performed in a continuous mode in the range of 10°–80° 2θ with collection speed 2°/min. The measured range was divided into intervals for data collection with the width of 0.02°. Measured data were evaluated in the PDXL2 program using International Center for Diffraction Data (ICDD) PDF-2 database, version 2013. Scanning electron microscopy (SEM) images of the prepared samples were obtained using scanning electron microscope JEOL JSM6510. Absorption spectrum of PpIX was measured in the range of 300–800 nm using dual beam UV–Vis spectrophotometer Varian Cary 100 with optical length of 1 cm. To evaluate luminescent properties of the prepared material, radioluminescence (RL), photoluminescence (PL) emission and decay curves were measured using the X-ray tube (RL, Seifert, 40 kV, 15 mA), steady state deuterium lamp (PL spectra) and nanosecond pulse nanoLED 339 nm and 389 nm (PL decays) as excitation sources. Detection part of the set-up was equipped with spectrofluorometer 5000M (Horiba Jobin Yvon), a single grating monochromator and photon counting detector TBX-04. RL and PL spectra were corrected for the spectral dependence of detection sensitivity. To detect the presence of singlet oxygen we used APF (aminophenyl fluorescein; Invitrogen™) commercial probe. 70 μL of APF was added into 2.5 mL of ethanol suspension containing 15 mg of ZnO:Ga@SiO2PpIX nanoparticles. X-ray tube with Cu anode (voltage 40 kV, current 30 mA, average wavelength Kα1,2 0.15418 nm) was used for irradiation. Samples were irradiated in polypropylene cuvette for 0–2 h. The dosimetry was performed using the Fricke dosimeter based on oxidation of Fe2+ to Fe3+. Absorbance measured at 303 nm, molar attenuation coefficient ε = 2164 dm3/mol/cm and radiochemical yield G (Fe3+) = 1.62 × 106 mol/J were used to evaluate the Fe3+ 14
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Fig. 2. SEM images of the as-prepared ZnO:Ga (A), ZnO:Ga@SiO2 (B) and ZnO:Ga@SiO2-PpIX (C).
Fig. 5. Normalized (to a maximum) PL spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX at RT excited at 340 nm as well as PL spectrum of and ZnO:Ga@SiO2-PpIX excited at 405 nm.
Fig. 3. Absorption and normalized PL spectra of PpIX at RT.
that when ZnO:Ga@SiO2-PpIX is excited into the ZnO:Ga core (at 339 nm). As mentioned earlier, the 635 nm band is associated with transition of monomers, while the 705 nm band is besides Q (0,1) transition also associated with formation of J-aggregates. The difference between emission excited directly (into the Soret band) with respect to that excited via energy transfer may be due to the fact that while direct excitation is absorbed mainly by monomers, the energy transfer preferably induces emission of aggregates. As a result, the emission from aggregates is stronger compared to that of monomers when excitation energy is delivered via energy transfer from the nanoparticle core. This fact is further supported by appearance of faster component in the luminescence decay (cf. Fig. 7b below). The difference in emission excited directly and that excited via energy transfer may also indicate that the energy transfer between the core and PpIX is nonradiative (such as FRET), since selection rules for aggregates vs monomers may be different due to different orientation of transition dipole moments with respect to the nanoparticle core. In case of radiative transfer one would expect qualitatively the same emission spectra as obtained under excitation into the Soret band, since both excitations correspond to a photon absorption. In Fig. 6 we present the PL decays of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX measured under excitation of the ZnO:Ga core, monitoring its excitonic emission. The excitonic emission is very fast, around 200 ps, which is quite close to the width of the excitation pulse (however, the value of 210 ps was previously reported for ZnO:Ga samples prepared in the same way (Procházková et al., 2015)). That is why with currently used apparatus it is difficult to reliably assess the exact value of the decay time as well as possible decay shortening expected under the presence of nonradiative transfer between the core and PpIX outer layer in the ZnO:Ga@SiO2-PpIX nanocomposite with respect to the ZnO:Ga core. On the other hand, the results reported above indicate that the nonradiative energy transfer mechanism seems more likely than that of radiative. There is also somewhat slower decay component of unknown origin observed in all decays whose intensity is, however, insignificant compared to that of the fast subnanosecond component. In Fig. 7 we also present the decays of the ZnO:Ga@SiO2-
Fig. 4. Radioluminescence spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@ SiO2-PpIX at RT. The spectrum of ZnO:Ga@SiO2-PpIX is multiplied by 10 for better visualization.
can see that SiO2 coating preserves the shape of the excitonic emission with no serious intensity losses with respect to that of the ZnO:Ga core. In contrast, after functionalization this emission in ZnO:Ga@SiO2-PpIX is almost completely suppressed. This observation is a strong indication of efficient energy transfer between the nanoparticle core and the PpIX outer layer in the functionalized sample. Normalized photoluminescence spectra are presented in Fig. 5. They were measured under excitation of ZnO:Ga core (339 nm) for all samples and also under excitation into the Soret band (405 nm) of PpIX in the ZnO:Ga@SiO2-PpIX sample. The band located around 390 nm is attributed to the excitonic emission, whose shape was not affected by the SiO2 coating. In case of functionalized ZnO:Ga@SiO2-PpIX, the excitonic emission is strongly suppressed in favour of PpIX emission that appears in the red spectral region. This is another strong indication of an energy transfer between the ZnO:Ga core and PpIX outer layer. Emission of PpIX in this sample is confirmed under direct excitation of PpIX into the Soret band. It is interesting to note, that the relative intensity of the two characteristic PpIX bands is exchanged compared to
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Fig. 6. PL decays of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX at RT. Excitation and emission wavelengths are reported in the figure. Empty circles are experimental data, dashed line represents the excitation pulse and the solid line is the fit of the function I(t) reported in the figure to the data.
PpIX nanocomposite where we monitor the 630 nm emission of PpIX. When excited directly into the Soret band, clean single exponential decay of PpIX with the decay time of 13.2 ns is observed, which is in a good agreement with the reported value (Patwari et al., 2017). When excited into the ZnO:Ga core, the same decay of PpIX is also observed. This is another evidence of the energy transfer between the core and the PpIX outer layer. There is also a faster component of the decay under the core excitation, of about 2 ns which may possibly be attributed to the PpIX J-aggregates (mentioned above). The ability of the synthesized nanocomposite ZnO:Ga@SiO2-PpIX to generate the singlet oxygen was tested by the APF chemical probe, which is sensitive to the presence of singlet oxygen. The results are reported in Fig. 8. X-ray irradiated sample exhibits an increase of the luminescence band observed around 530 nm under the increase of the X-ray dose. This band appears due to the presence of 1O2 or other reactive oxygen species (Price et al., 2009). Broad bands observed in the region above 600 nm are due to emission of PpIX. The desired effect is not too strong which calls for further optimization of nanocomposite synthesis. There is certainly room for improvements in the processes of SiO2 coating and functionalization. Another possible way appears in modification of ZnO:Ga core. Tuning of the ZnO:Ga excitonic emission by doping with Mg or Cd ions was already mentioned in (Čuba et al., 2014). As displayed in Fig. 9, ZnCdO:Ga nanoparticles with 7% or 12%
Fig. 8. RT PL spectra of ZnO:Ga@SiO2-PpIX + APF before and after X-ray irradiation for 30 and 115 min. The irradiation dose is 34 Gy/h (for details see section 2.2). In the inset the detail of the 530 nm band.
Cd doping manifest the red shift of the excitonic emission in the RL spectra with significantly improved match with the Soret band of PpIX. This improvement would enhance the efficiency of energy transfer between the core and photosensitizer and consequently also the singlet oxygen production.
Fig. 7. PL decays of ZnO:Ga@SiO2-PpIX at RT. Excitation and emission wavelengths are reported in the figure. Empty circles are experimental data, dashed line represents the excitation pulse and the solid line is the fit of the function I(t) reported in the figure to the data. 16
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Fig. 9. Absorption spectra of PpIX and normalized RL spectra of ZnO:Ga, ZnCdO:Ga (7% Cd) and ZnCdO:Ga (12% Cd) at RT.
4. Conclusion A nanocomposite based on the scintillating ZnO:Ga core, potentially suitable for PDTX application, was successfully synthesized. The core was prepared by radiation induced method, subsequently coated by SiO2 shell using a modified sol-gel process. The SiO2 layer facilitates the process of functionalization by PpIX molecule serving as a photosensitizer. Combined results of steady state and time resolved spectroscopy suggest the presence of an energy transfer between the ZnO:Ga core and PpIX outer layer. In particular, i) emission of the core is almost completely suppressed in the RL spectrum of functionalized particle, ii) PL spectrum features characteristic emission of PpIX in the red spectral region under the core excitation, iii) PL decay features PpIX decay under the core excitation. The results indicate that the mechanism of energy transfer is more likely to be nonradiative. The ability of synthesized ZnO:Ga@SiO2-PpIX nanocomposite to produce the singlet oxygen was demonstrated with the help of the chemical APF probe. Despite indications that there is still room for an improvement of nanocomposite synthesis, it seems evident that the tested nanocomposite represents a promising candidate for PDTX application. Acknowledgements Financial support of the Czech Science Foundation under Grant GA 17-06479S is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.radmeas.2018.12.008. References Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn, S.M., Hamblin, M.R., Juzeniene, A., Kessel, D., et al., 2011. Photodynamic therapy of cancer: an update. CA A Cancer J. Clin. 61, 250–281. Bárta, J., Čuba, V., Pospíšil, M., Jarý, V., Nikl, M., 2012. Radiation-induced preparation of pure and Ce-doped lutetium aluminium garnet and its luminescent properties. J. Mater. Chem. 22, 16590–16597. Bulin, A.-L., Truillet, C., Chouikrat, R., Lux, F., Frochot, C., Amans, D., Ledoux, G., Tillement, O., Perriat, P., Barberi-Heyob, M., Dujadin, C., 2013. X-ray-induced singlet oxygen activation with nanoscintillator-coupled porphyrins. J. Phys. Chem. C 117, 21583–21589. Chen, W., 2008. Nanoparticle self-lighting photodynamic therapy for cancer treatment. J. Biomed. Nanotechnol. 4, 369–376. Chen, W., Zhang, J., 2006. Using nanoparticles to enable simultaneous radiation and photodynamic therapies for cancer treatment. J. Nanosci. Nanotechnol. 6, 1159–1166. Chen, G., Agren, H., Ohulchansky, T.Y., Prasad, P.N., 2015. Light upconverting core–shell nanostructures: nanophotonic control for emerging applications. Chem. Soc. Rev. 44, 1680–1713. Čuba, V., Procházková, L., Bárta, J., Vondrášková, A., Pavelková, T., Mihóková, E., Jarý,
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Contents lists available at ScienceDirect
Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Novel scintillating nanocomposite for X-ray induced photodynamic therapy Lenka Procházková a b c
, Iveta Terezie Pelikánová , Eva Mihóková , Roman Dědic , Václav Čuba
a,b
b
a,∗
c
T
b
Institute of Physics of the Czech Academy Od Sciences, Cukrovarnická 10, 162 53, Prague, Czech Republic Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7, 115 19, Prague, Czech Republic Charles University, Faculty of Math and Physics, Ke Karlovu 3, 121 16, Prague, Czech Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanocomposite Photodynamic therapy Singlet oxygen Zinc oxide Scintillator
We synthesize a novel prospective nanocomposite material for X-ray induced photodynamic therapy. ZnO:Ga core is coated by the SiO2 shell facilitating a functionalization by a photosensitizer, protoporphyrin PpIX layer. By steady-state and time resolved spectroscopy we confirm the presence of energy transfer between the core and the photosensitizer, an essential requirement to induce a cytotoxic effect. Using the commercial chemical probe we also demonstrate the ability of prepared ZnO:Ga@SiO2-PpIX nanocomposite to generate singlet oxygen. Presented results show that this nanocomposite can be considered as a good candidate for potential application in X-ray induced photodynamic therapy.
1. Introduction Photodynamic therapy (PDT) is treatment in which visible or nearinfrared light is used to activate a photosensitizer (PS), administered to target a diseased tissue of interest. In the presence of ground state oxygen, the light-activated PS creates reactive oxygen species, especially highly cytotoxic singlet oxygen, that can induce tissue death (Henderson and Dougherty, 1992; Agostinis et al., 2011). PDT was developed primarily for treatment of cancer and precancers. Due to limited depth of light penetration, PDT is most commonly used to treat superficial lesions, like cancer or precancer of the skin (MacCormack, 2006). To overcome the problem of reaching deeply residing tumors in conventional PDT, several approaches have been developed. One or more optical fibers for light delivery are inserted into the target tissue (Shafirstein et al., 2017). Other approaches use specific types of nanoparticles to which PS is attached. Two-photon excitation PDT exploits upconversion nanoparticles that are excited by two-photon absorption of near infrared light and emit higher energy photons to activate PS molecules (Yang et al., 2015; Park et al., 2015; Idris et al., 2015; Chen et al., 2015; Shanmugam et al., 2014; Zhou et al., 2012). In SelfLighting PDT (Chen, 2008; Zhang et al., 2008) the light is generated by afterglow nanoparticles with attached photosensitizers. When the nanoparticle-photosensitizer conjugates are targeted to tumor, the light from afterglow nanoparticles will activate the photosensitizers. Therefore, no external light is required for treatment. X-ray induced PDT (PDTX) exploits X-ray excited photosensitizers (Ma et al., 2014; Liu et al., 2017), quantum dots (Juzenas et al., 2008)
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or scintillating nanoparticles (Chen and Zhang, 2006; Wang et al., 2016). PDTX is a combination of radiotherapy and PDT providing more efficient tumor cell killing than the radiotherapy alone. After the X-ray energy is absorbed by a scintillating nanoparticle, it is either radiatively or nonradiatively transferred to the PS molecule. To achieve a cytotoxic effect at therapeutic doses, several parameters, such as light yield of a scintillating nanoparticle, efficiency of energy transfer to PS and cellular uptake of nanoparticles have to be well optimized (Morgan et al., 2009). The nanoparticle uptake by tumor blood vessels can be improved by enhanced permeability and retention (EPR) effect promoted, among others, by suitable nanoparticle size of about 50 nm (Maeda et al., 2013). Various nanocomposite systems have already been shown as potential candidates for PDTX treatment. These systems were based on PS molecule attached to a scintillating core, such as Y2O3 (Scaffidi et al., 2011), Tb2O3 (Bulin et al., 2013), LaF3:Ce3+ (Zou et al., 2014), CeF3:Tb3+ (Popovich et al., 2016), LuAG:Pr3+ (Popovich et al., 2018) or ZnO (Liu et al., 2008). ZnO is a known scintillator with ultrafast, subnanosecond excitonic decay. It is a semiconductor with a wide band gap of 3.4 eV. Its extremely high exciton binding energy of about 60 meV ensures efficient luminescence at room temperature (RT). ZnO in various forms has been studied due to a number of applications especially in optoelectronics. Controlled doping with Ga3+ ions together with heat treatment in reducing atmosphere suppress the slow defect emission around 500 nm and enhances the excitonic emission around 400 nm (Procházková et al., 2015). Therefore, ZnO:Ga3+ was recently also studied for applications requiring fast timing (Turtos et al., 2016), such as time-of
Corresponding author. E-mail address: [email protected] (E. Mihóková).
https://doi.org/10.1016/j.radmeas.2018.12.008 Received 15 October 2018; Received in revised form 11 December 2018; Accepted 14 December 2018 Available online 17 December 2018 1350-4487/ © 2018 Elsevier Ltd. All rights reserved.
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flight positron emission tomography (Karp et al., 2008; Jones and Townsend, 2017; Gundacker et al., 2016). In this work we design a novel nanocomposite system as a candidate for PDTX application. The nanocomposite consists of the ZnO:Ga scintillating core coated by the SiO2 shell facilitating its conjugation to protoporphyrin IX (PpIX) serving as a photosensitizer. We synthesize this system, study its luminescence and scintillation properties to test the energy transfer between the ZnO:Ga3+ core and PpIX outer layer and we also show its ability to generate the singlet oxygen. 2. Materials and methods 2.1. Nanocomposite synthesis ZnO:Ga core was synthesized by photo-induced method (Bárta et al., 2012) using the following chemicals: zinc oxide, gallium nitrate, (all chemicals from Aldrich, 99.999% trace metals basis), formic acid, nitric acid (Penta, p.a.) and hydrogen peroxide (Penta, p.a.). After the irradiation of aqueous solutions, formed solid zinc peroxide was separated and decomposed at 250 °C in air. Two-step annealing followed: annealing at 1000 °C in air performed in the Clasic 0415 VAC vacuum furnace and additional annealing at 800 °C using the Setaram LabSys Evo thermoanalyzer under the Ar/H2 (20:1) atmosphere. The prepared ZnO:Ga nanopowder was coated by a silica layer using a modified sol-gel process described in (Liu et al., 1998), for more details, see (Popovich et al., 2018). Obtained silica-coated ZnO:Ga nanoparticles were conjugated with PpIX using a modified functionalization procedure reported in (Nowostawska et al., 2011), for more details, see (Popovich et al., 2018).
Fig. 1. Diffraction patterns of synthesized nanoparticles (indicated in the figure) compared with the standard dat of ZnO:Ga from ICDD PDF-2 database (card No. 01-073-1368, dashed lines in the figure). Data for ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX are vertically shifted for better visualization.
concentration c (Fe3+). The radiation dose rate D is calculated from c (Fe3+) - c0(Fe3+) = ρ × G (Fe3+) × D, where ρ = 1.024 kg/m3. The obtained value 34 Gy/h for X-ray source used reflects the dose absorbed by the solutions in plastic cuvettes, which may slightly differ in the case of suspensions used in real experiments. 3. Results The diffraction patterns of all synthesized materials are shown in Fig. 1. They are all in agreement with that of ZnO:Ga standard (dashed lines, card No. 01-073-1368) from ICDD PDF-2 database. Prepared nanoparticles correspond to hexagonal crystal structure of wurtzite. The average crystallite size is about 300 nm. This size was determined by application of Halder-Wagner method using FWHM (full width at half maximum) of peaks and the Scherrer constant K = 0.94. The absence of additional peaks in diffraction patterns excludes the presence of any other crystalline phase. Furthermore, it confirms that coating of ZnO:Ga nanoparticle by SiO2 layer and subsequent functionalization preserve the crystal structure of the nanoparticle as well as the fact that SiO2 layer is amorphous. In Fig. 2 we present SEM images of all prepared nanoparticles. The images show ZnO:Ga grains (A), ZnO:Ga modified by the surface SiO2 layer (B) and further functionalized by PpIX (C). As mentioned above, cytotoxic effects of prepared functionalized nanoparticles ZnO:Ga@SiO2-PpIX can only be achieved if there exists an efficient energy transfer between the ZnO:Ga core and PpIX outer layer. A prerequisite for such energy transfer, either radiative or nonradiative, is an overlap between the absorption spectrum of PpIX and RL spectrum of ZnO:Ga. In Fig. 3a we show absorption spectrum of PpIX featuring a strong absorption band peaking around 400 nm, so called Soret band, and weaker Q-bands peaking between 500 and 650 nm. Typical PL spectrum of PpIX under excitation into the Soret band is also shown. Its two bands are peaking at about 635 nm and 705 nm. The peak around 635 nm is due to zero-phonon Q (0,0) emission of monomeric PpIX (Hope et al., 2016; Scolaro et al., 2002). The 705 nm peak occurs via Q (0,1) transition known as J-band, which is also associated with the result of formation of J-aggregates (Villari et al., 2012) when the porphyrin molecules self-aggregate (Hope et al., 2016; Scolaro et al., 2002). Comparison to PpIX absorption with the ZnO:Ga RL spectrum displayed in Figs. 4 and 9 confirms a significant overlap between the Soret band of PpIX and ZnO:Ga RL spectrum. Radioluminescence spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@ SiO2-PpIX are presented in Fig. 4. ZnO:Ga nanoparticles feature an intense emission band around 390 nm attributed to emission of MottWannier exciton formed under excitation of the ZnO host lattice. One
2.2. Characterization methods The XRD measurements were performed using Rigaku MiniFlex 600 diffractometer equipped with Cu X-ray tube (average wavelength Kα1,20.15418 nm). High voltage and current settings used were 40 kV and 15 mA, respectively. The measurement was performed in a continuous mode in the range of 10°–80° 2θ with collection speed 2°/min. The measured range was divided into intervals for data collection with the width of 0.02°. Measured data were evaluated in the PDXL2 program using International Center for Diffraction Data (ICDD) PDF-2 database, version 2013. Scanning electron microscopy (SEM) images of the prepared samples were obtained using scanning electron microscope JEOL JSM6510. Absorption spectrum of PpIX was measured in the range of 300–800 nm using dual beam UV–Vis spectrophotometer Varian Cary 100 with optical length of 1 cm. To evaluate luminescent properties of the prepared material, radioluminescence (RL), photoluminescence (PL) emission and decay curves were measured using the X-ray tube (RL, Seifert, 40 kV, 15 mA), steady state deuterium lamp (PL spectra) and nanosecond pulse nanoLED 339 nm and 389 nm (PL decays) as excitation sources. Detection part of the set-up was equipped with spectrofluorometer 5000M (Horiba Jobin Yvon), a single grating monochromator and photon counting detector TBX-04. RL and PL spectra were corrected for the spectral dependence of detection sensitivity. To detect the presence of singlet oxygen we used APF (aminophenyl fluorescein; Invitrogen™) commercial probe. 70 μL of APF was added into 2.5 mL of ethanol suspension containing 15 mg of ZnO:Ga@SiO2PpIX nanoparticles. X-ray tube with Cu anode (voltage 40 kV, current 30 mA, average wavelength Kα1,2 0.15418 nm) was used for irradiation. Samples were irradiated in polypropylene cuvette for 0–2 h. The dosimetry was performed using the Fricke dosimeter based on oxidation of Fe2+ to Fe3+. Absorbance measured at 303 nm, molar attenuation coefficient ε = 2164 dm3/mol/cm and radiochemical yield G (Fe3+) = 1.62 × 106 mol/J were used to evaluate the Fe3+ 14
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Fig. 2. SEM images of the as-prepared ZnO:Ga (A), ZnO:Ga@SiO2 (B) and ZnO:Ga@SiO2-PpIX (C).
Fig. 5. Normalized (to a maximum) PL spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX at RT excited at 340 nm as well as PL spectrum of and ZnO:Ga@SiO2-PpIX excited at 405 nm.
Fig. 3. Absorption and normalized PL spectra of PpIX at RT.
that when ZnO:Ga@SiO2-PpIX is excited into the ZnO:Ga core (at 339 nm). As mentioned earlier, the 635 nm band is associated with transition of monomers, while the 705 nm band is besides Q (0,1) transition also associated with formation of J-aggregates. The difference between emission excited directly (into the Soret band) with respect to that excited via energy transfer may be due to the fact that while direct excitation is absorbed mainly by monomers, the energy transfer preferably induces emission of aggregates. As a result, the emission from aggregates is stronger compared to that of monomers when excitation energy is delivered via energy transfer from the nanoparticle core. This fact is further supported by appearance of faster component in the luminescence decay (cf. Fig. 7b below). The difference in emission excited directly and that excited via energy transfer may also indicate that the energy transfer between the core and PpIX is nonradiative (such as FRET), since selection rules for aggregates vs monomers may be different due to different orientation of transition dipole moments with respect to the nanoparticle core. In case of radiative transfer one would expect qualitatively the same emission spectra as obtained under excitation into the Soret band, since both excitations correspond to a photon absorption. In Fig. 6 we present the PL decays of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX measured under excitation of the ZnO:Ga core, monitoring its excitonic emission. The excitonic emission is very fast, around 200 ps, which is quite close to the width of the excitation pulse (however, the value of 210 ps was previously reported for ZnO:Ga samples prepared in the same way (Procházková et al., 2015)). That is why with currently used apparatus it is difficult to reliably assess the exact value of the decay time as well as possible decay shortening expected under the presence of nonradiative transfer between the core and PpIX outer layer in the ZnO:Ga@SiO2-PpIX nanocomposite with respect to the ZnO:Ga core. On the other hand, the results reported above indicate that the nonradiative energy transfer mechanism seems more likely than that of radiative. There is also somewhat slower decay component of unknown origin observed in all decays whose intensity is, however, insignificant compared to that of the fast subnanosecond component. In Fig. 7 we also present the decays of the ZnO:Ga@SiO2-
Fig. 4. Radioluminescence spectra of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@ SiO2-PpIX at RT. The spectrum of ZnO:Ga@SiO2-PpIX is multiplied by 10 for better visualization.
can see that SiO2 coating preserves the shape of the excitonic emission with no serious intensity losses with respect to that of the ZnO:Ga core. In contrast, after functionalization this emission in ZnO:Ga@SiO2-PpIX is almost completely suppressed. This observation is a strong indication of efficient energy transfer between the nanoparticle core and the PpIX outer layer in the functionalized sample. Normalized photoluminescence spectra are presented in Fig. 5. They were measured under excitation of ZnO:Ga core (339 nm) for all samples and also under excitation into the Soret band (405 nm) of PpIX in the ZnO:Ga@SiO2-PpIX sample. The band located around 390 nm is attributed to the excitonic emission, whose shape was not affected by the SiO2 coating. In case of functionalized ZnO:Ga@SiO2-PpIX, the excitonic emission is strongly suppressed in favour of PpIX emission that appears in the red spectral region. This is another strong indication of an energy transfer between the ZnO:Ga core and PpIX outer layer. Emission of PpIX in this sample is confirmed under direct excitation of PpIX into the Soret band. It is interesting to note, that the relative intensity of the two characteristic PpIX bands is exchanged compared to
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Fig. 6. PL decays of ZnO:Ga, ZnO:Ga@SiO2 and ZnO:Ga@SiO2-PpIX at RT. Excitation and emission wavelengths are reported in the figure. Empty circles are experimental data, dashed line represents the excitation pulse and the solid line is the fit of the function I(t) reported in the figure to the data.
PpIX nanocomposite where we monitor the 630 nm emission of PpIX. When excited directly into the Soret band, clean single exponential decay of PpIX with the decay time of 13.2 ns is observed, which is in a good agreement with the reported value (Patwari et al., 2017). When excited into the ZnO:Ga core, the same decay of PpIX is also observed. This is another evidence of the energy transfer between the core and the PpIX outer layer. There is also a faster component of the decay under the core excitation, of about 2 ns which may possibly be attributed to the PpIX J-aggregates (mentioned above). The ability of the synthesized nanocomposite ZnO:Ga@SiO2-PpIX to generate the singlet oxygen was tested by the APF chemical probe, which is sensitive to the presence of singlet oxygen. The results are reported in Fig. 8. X-ray irradiated sample exhibits an increase of the luminescence band observed around 530 nm under the increase of the X-ray dose. This band appears due to the presence of 1O2 or other reactive oxygen species (Price et al., 2009). Broad bands observed in the region above 600 nm are due to emission of PpIX. The desired effect is not too strong which calls for further optimization of nanocomposite synthesis. There is certainly room for improvements in the processes of SiO2 coating and functionalization. Another possible way appears in modification of ZnO:Ga core. Tuning of the ZnO:Ga excitonic emission by doping with Mg or Cd ions was already mentioned in (Čuba et al., 2014). As displayed in Fig. 9, ZnCdO:Ga nanoparticles with 7% or 12%
Fig. 8. RT PL spectra of ZnO:Ga@SiO2-PpIX + APF before and after X-ray irradiation for 30 and 115 min. The irradiation dose is 34 Gy/h (for details see section 2.2). In the inset the detail of the 530 nm band.
Cd doping manifest the red shift of the excitonic emission in the RL spectra with significantly improved match with the Soret band of PpIX. This improvement would enhance the efficiency of energy transfer between the core and photosensitizer and consequently also the singlet oxygen production.
Fig. 7. PL decays of ZnO:Ga@SiO2-PpIX at RT. Excitation and emission wavelengths are reported in the figure. Empty circles are experimental data, dashed line represents the excitation pulse and the solid line is the fit of the function I(t) reported in the figure to the data. 16
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Fig. 9. Absorption spectra of PpIX and normalized RL spectra of ZnO:Ga, ZnCdO:Ga (7% Cd) and ZnCdO:Ga (12% Cd) at RT.
4. Conclusion A nanocomposite based on the scintillating ZnO:Ga core, potentially suitable for PDTX application, was successfully synthesized. The core was prepared by radiation induced method, subsequently coated by SiO2 shell using a modified sol-gel process. The SiO2 layer facilitates the process of functionalization by PpIX molecule serving as a photosensitizer. Combined results of steady state and time resolved spectroscopy suggest the presence of an energy transfer between the ZnO:Ga core and PpIX outer layer. In particular, i) emission of the core is almost completely suppressed in the RL spectrum of functionalized particle, ii) PL spectrum features characteristic emission of PpIX in the red spectral region under the core excitation, iii) PL decay features PpIX decay under the core excitation. The results indicate that the mechanism of energy transfer is more likely to be nonradiative. The ability of synthesized ZnO:Ga@SiO2-PpIX nanocomposite to produce the singlet oxygen was demonstrated with the help of the chemical APF probe. Despite indications that there is still room for an improvement of nanocomposite synthesis, it seems evident that the tested nanocomposite represents a promising candidate for PDTX application. Acknowledgements Financial support of the Czech Science Foundation under Grant GA 17-06479S is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.radmeas.2018.12.008. References Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn, S.M., Hamblin, M.R., Juzeniene, A., Kessel, D., et al., 2011. Photodynamic therapy of cancer: an update. CA A Cancer J. Clin. 61, 250–281. Bárta, J., Čuba, V., Pospíšil, M., Jarý, V., Nikl, M., 2012. Radiation-induced preparation of pure and Ce-doped lutetium aluminium garnet and its luminescent properties. J. Mater. Chem. 22, 16590–16597. Bulin, A.-L., Truillet, C., Chouikrat, R., Lux, F., Frochot, C., Amans, D., Ledoux, G., Tillement, O., Perriat, P., Barberi-Heyob, M., Dujadin, C., 2013. X-ray-induced singlet oxygen activation with nanoscintillator-coupled porphyrins. J. Phys. Chem. C 117, 21583–21589. Chen, W., 2008. Nanoparticle self-lighting photodynamic therapy for cancer treatment. J. Biomed. Nanotechnol. 4, 369–376. Chen, W., Zhang, J., 2006. Using nanoparticles to enable simultaneous radiation and photodynamic therapies for cancer treatment. J. Nanosci. Nanotechnol. 6, 1159–1166. Chen, G., Agren, H., Ohulchansky, T.Y., Prasad, P.N., 2015. Light upconverting core–shell nanostructures: nanophotonic control for emerging applications. Chem. Soc. Rev. 44, 1680–1713. Čuba, V., Procházková, L., Bárta, J., Vondrášková, A., Pavelková, T., Mihóková, E., Jarý,
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